Enzymatic reactions exhibit remarkable selectivity and efficiency, the likes of which are rarely achieved in bench-top chemical reactions. While it is clear that the biochemical prowess of an enzyme arises from its highly-ordered structure, the detailed mechanism by which it functions has proven elusive. This is because enzymes are not simply static macromolecules that host an active site, as depicted by their crystal structure; rather, they are dynamic molecules whose choreographed motions can gate the transport of substrate to and from the active site and can modulate over time the activity of that site. To develop a mechanistic understanding into how enzymes function, it is essential to study this choreography of life at the atomic level with ultrafast time resolution. We investigate this choreography of life using time-resolved techniques based on the pump-probe method. The pump is often a laser pulse that triggers at a well-defined instant of time the process we wish to investigate. The duration of the laser pulse can be as short as a few tens of femtoseconds, which allows us to access the chemical time scale for molecular motion. After laser excitation, a time-delayed probe pulse is directed through the pump-illuminated volume to interrogate the system. When the probe pulse is derived from an X-ray source, we can probe the protein structure via Laue diffraction or Small- and Wide-Angle-X-ray-Scattering (SAXS/WAXS). When the probe pulse is generated in the uv-vis or mid-IR region, we can interrogate the system spectroscopically. The time resolution of the pump-probe method is limited only by the duration of the pump and probe pulses and the timing jitter between them. Each pump-probe measurement produces a time-resolved snapshot of the protein. By stitching together a series of snapshots, we create a movie that, in the case of time-resolved Laue diffraction, provides a near-atomic view of the correlated structure changes triggered by the pump pulse. We can literally watch a protein as it functions! In time-resolved NMR studies, a rapid pressure jump triggers a change in the protein structure, the dynamics of which can be probed with side chain specificity. Our efforts to develop time-resolved methods suitable for investigating biomolecule dynamics and function are summarized here. The Ultrafast Biophysical Chemistry Section of LCP maintains a laser lab in Bldg. 5, Rm. B2-10 with numerous operational laser systems including two femtosecond regenerative amplifier systems, associated home-built Optical Parametric Amplifiers (OPAs), a nanosecond Optical Parametric Oscillator (OPO), and several frequency-doubled Nd:YAG lasers. The OPAs and OPO are capable of producing intense optical pulses over a broad spectral range spanning the uv to the mid-IR. When the pump and probe pulses used to photoexcite and monitor samples are generated with OPAs, the time resolution of the spectroscopic measurement can be less than 100 femtoseconds, and the relative time of arrival can be varied from femtoseconds to a few nanoseconds via an optical delay line. An Optical Parametric Oscillator (OPO) with broad tunability throughout the visible generates 2-3 ns pump pulses that can be delayed electronically out to seconds. A Q-switched, frequency-doubled Nd:YAG laser generates 200-ns pulses at 532 nm that can be delayed electronically out to seconds. A 527-nm CW laser can be electronically gated on and off with an acousto-optic modulator (AOM) capable of approximately 200 ns switching times. These sources can be used independently or in conjunction with one another. The electronic synchronization capabilities needed to effectively operate this array of laser systems is beyond the capacity of our existing electronic timing system. Hence we are developing a third-generation Suzaku Field-Programmable-Gate-Array (FPGA) based timing system capable of controlling independently the relative timing of up to 24 digital outputs, and are implementing a novel player-piano paradigm for gating the FPGA-generated pulses. With this approach, data acquisition sequences appropriate for experiments ranging from time-resolved spectroscopy in our laser lab to time-resolved Laue crystallography or time-resolved SAXS/WAXS studies on the BioCARS beamline at the APS are executed by playing repeating pulse patterns that correspond to measures of music. Briefly, the Suzaku board consists of a Xilinx FPGA chip and its associated micro-Linux processor. To implement player piano mode operation, we developed interrupt handler code capable of responding to interrupts at a rate of 1 kHz without missing a beat. The pulse sequences required for all output channels are coded in one measure-long packets, each of which fully specifies the repeating pattern required for all output channels. These packets are generated, indexed, and stored in RAM. The interrupt handler is fed a sequence of indices that specify the order in which the packets are played, and therefore defines the data acquisition sequence in a deterministic fashion. We have built four identical FPGA boards so we can employ this deterministic approach to data acquisition in time-resolved studies conducted in various locations, including our femtosecond laser lab and the BioCARS x-ray beamline. We continue to refine the time-resolved X-ray infrastructure we developed on the BioCARS Beamline at the APS. Briefly, this development effort began in FY2005 when Dr. Marvin Gershengorn, then Director of Intramural Research at NIDDK, committed >$1M to procure the capital equipment needed. The major components acquired/developed include a high-powered laser system, two new undulators, and chopper/shutter systems used to isolate individual x-ray pulses. After replacing the existing U33 undulator (33-mm magnetic period) with two new short-period inline U23 and U27 undulators, BioCARS produced one of the hottest x-ray beams on the planet. More recently, we upgraded the BioCARS beamline by incorporating a secondary K-B mirror pair, a new high-speed diffractometer, and a new $1.6 M high-speed X-ray detector. The high-speed diffractometer is designed to take full advantage of the tighter x-ray focus and the high-speed detector. Thanks to the relatively small spot size that can be generated with the secondary K-B mirror pair, it is possible to focus a 1 mJ infrared pulse down to a dimension small enough to heat samples in a glass capillary by more than 10 degrees Celsius. Using a high-precision temperature controller to set the temperature just below a protein's unfolding temperature, this magnitude T-jump is sufficient to trigger unfolding of a protein, and allow us to investigate the dynamics of protein unfolding with unprecedented spatial resolution. The time-resolution achieved is currently limited by the duration of the infrared laser pulse, which is about 5 ns. In a collaboration with Ad Bax, we developed a novel high-pressure apparatus capable of rapidly switching the hydrostatic pressure in a zirconia NMR tube between 1 bar and up to 3 kbar, with the transition requiring only a few milliseconds. Many proteins spontaneously unfold at high pressure, or can be engineered to do so via mutagenesis. Thus, rapid changes in pressure can trigger protein unfolding or folding. By acquiring 2D and 3D NMR spectra over a series of time delays following the high-pressure transition, the folding/unfolding pathway can be unveiled at an unprecedented level of detail. The first published application of this novel methodology was a study of the folding time required to protect side chains of ubiquitin from hydrogen exchange.